RNAi therapeutics run interference

BY CHRISTEN BROWNLEE

RNA interference is getting the axe at major pharmaceutical companies. Is it too soon?

In February of 2008, the future looked bright for Arthur Krieg and a little technology known as RNA interference. Krieg had just joined Pfizer after the pharmaceutical giant acquired Coley Pharmaceuticals, a company he had cofounded in 1997 and where he had served as the chief scientific officer and executive vice president of research and development. Based on his extensive experience in developing oligodeoxynucleotides, Pfizer asked Krieg to head up a new division devoted solely to RNAi.

"I had to tell the group that we were being shut down. All the sites associated with our RNAi program were being closed, and all positions were being eliminated."

This biological phenomenon, which knocks down or dramatically lowers the protein output of selected genes by inserting a piece of double-stranded RNA into cells, was making leaps and bounds in the lab. Pfizer and many other big pharma companies saw this new technology as the wave of the future – a new way to target practically any gene that has a known sequence.

But this February – nearly three years to the day after he started his new position – Krieg stood in front of the 100-member group that Pfizer had hired to run its RNAi program to make a terrible announcement.

“I had to tell the group that we were being shut down. All the sites associated with our RNAi program were being closed, and all positions were being eliminated,” he says.

What happened in those three intervening years? Whatever it was had also happened to other big pharma companies who invested in RNAi. Roche, Novartis, and Abbott Labs recently terminated their RNAi programs and severed their ties with biotech partners who were helping to develop the technology, sending shock waves through the field.

But while RNAi is getting the axe at big pharma, the technology has continued to march quietly forward at smaller biotech companies and academic labs. Animal studies recently have shown that RNAi could hold incredible promise for treating HIV, and human studies for cancers and other diseases are moving ahead into Phase 1 and 2 clinical trials. Could big pharma have pulled the plug before RNAi hit the big time?

Pulling back

RNAi still is an incredibly nascent technology. It only was discovered in the late 1990s, detailed for the first time in a seminal 1998 Nature paper by Andrew Fire and Craig Mello. There, the two scientists wrote about a strange phenomenon whereby double-stranded RNA injected into C. elegans had the power to potently silence genes. Since then, researchers have discovered that this concept also works for all living things – plants, insects and, eventually, humans.

The ability to silence any gene in the body would prove to be an incredible boon to research. No longer would scientists patiently have to wait a year or longer to make knockout mice to study gene function – with RNAi, the knockout could happen instantly. Additionally, trying to decipher the functions of genes essential to life wouldn’t necessarily have to happen at the embryonic stage, before organisms bit the dust early on. Rather, researchers could knock these down later in life and see what happens.

RNAi therapeutics start acting when a short piece of double-stranded RNA (somewhere between 20 and 25 or so nucleotides) enters a cell. In the cytoplasm, the RNA bumps into an enzyme called dicer, which acts like a samurai sword-wielding ninja, chopping the dsRNA into smaller pieces known as small interfering RNAs. These siRNA unzip into two strands. One of the strands gets picked up by a group of different proteins known as the RNA-induced silencing complex. The entire package hunts down strands of messenger RNA inside a cell that complement the contained siRNA. Once that complementary strand is found, a group of enzymes chop up the matching mRNA. Without that mRNA, the corresponding protein can’t get made. Since most diseases are the result of problematic proteins – either faulty construction of a necessary protein or too much of a good thing – ridding cells of certain proteins might lessen their consequences or, in the case of some infectious diseases, cure certain conditions altogether. Figure from Robinson, R. (2004) PLoS Biology 2, e28.

After Fire and Mello’s influential paper, basic researchers flocked to RNAi. It was enough to win the two researchers the 2006 Nobel Prize in Physiology or Medicine, an unheard of turnaround in a novel field.

But years before Fire and Mello accepted their prize in Stockholm, RNAi also had caught the notice of pharmaceutical companies. The ability to silence specific genes, thereby ridding the body of pesky disease-causing proteins, also could provide unprecedented gains for therapies. Though about two-thirds of pharmaceutical targets currently are considered undruggable – with no small molecule currently identified or no way to specifically hit a target without causing other unspecific and undesirable effects – RNAi could provide a way to home in on a desired target through its gene sequence, making more targets druggable.

The problem, Krieg says, is that delivering double-stranded RNA has proven incredibly tricky. RNAs that are too long provoke an interferon response that muddies any effect of the RNAs themselves – a holdover from the earlier days of evolution when double-stranded RNA automatically equaled a viral attack. RNAs that are too short might not be enough to prompt sufficient interference. Naked RNAs are vulnerable to degrading RNAses circulating in blood and tissues. Finding a way to coat RNAs of the right size and sequence now has become a field in itself.

“People thought, ‘Here we have this platform in which we’re going to identify genes for breast cancer or chronic obstructive pulmonary disease or Alzheimer’s, and then we’ll have these RNAi compounds that we can give to patients, and they’ll go where we want them to go and the patients will get better,’” Krieg says. “With more experience working with this, they realized that it’s not going to work that way.”

Delivery seems to have proven tricky for other big pharma companies as well, even those that are sticking with the technology. Over e-mail, Alan Sachs, the global head of exploratory and translational science at Merck, noted that the company had explored more than 300 different delivery technologies for a range of disease targets. But although Merck acquired RNAi biotech Sirna Therapeutics in 2006 for the astronomical sum of $1.15 billion, the company has yet to have any RNAi therapeutic candidates in clinical trials.

“Merck recognized from the outset that developing RNAi therapeutics would be a long-term investment and not a quick path to blockbuster drugs,” he says, adding that the company “is taking a careful and steady approach to RNAi.”

But the length of that investment – the time it would take to understand the best targets and develop the most effective delivery strategies – may have been more than the companies that dropped RNAi could bear. Though Krieg and his colleagues at Pfizer hoped to get an RNAi compound into the pipeline by this year and were making progress with their top candidate, a treatment for liver cancer, the team still had some distance to go by the time their program folded.

Krieg suspects that the story at Pfizer is the same elsewhere. As the company started to realize how much of an outlay RNAi would take to get to the clinic, it realized it probably wouldn’t be able to recoup its investment. “The lifetime of patents by the time you get a drug approved is really insufficient to return the investment on a drug most of the time,” he explains. “If you look at the pipeline, it’s inadequate to support their infrastructure.”

Going strong

Roche, which also has invested heavily in RNAi therapeutics during the past few years, pouring hundreds of millions into its collaborations with biotech partners, issued a cagey statement about its own decision to leave RNAi behind. “The primary goal is to enable this important scientific work to continue outside of Roche and offer the best chance of success in providing benefits to patients,” it said, adding that “Roche would consider the possibility of re-entering the field through external collaboration as clinical stage compounds emerge.”

That re-entry might be just around the corner, says Barry Greene of Alnylam Pharmaceuticals, an RNAi therapeutics company that partnered with Roche until the company severed its agreement with Alnylam late last year.

Greene points out that Alnylam and other companies are rapidly moving ahead with their own RNAi therapeutics. At his company alone, several RNAi-based drugs already are moving through clinical trials. Alnylam even started its own initiative earlier this year named “Alnylam 5 x 15”—an effort to get five products in advanced clinical development by the year 2015. The most advanced therapeutic candidate in this program is a drug for transthyretin amyloidosis, an autosomal inherited disease that affects about 50,000 people worldwide and universally kills patients within five to 15 years of diagnosis. The drug currently is in Phase 2 clinical trials.

This disease, which attacks the liver, is an attractive target since the organ has a natural propensity to take up the nanolipid delivery vehicles created by Alnylam partner Tekmira that encapsulate the desired RNAi snippets.

Greene notes that Alnylam also has other RNAi-based drugs in clinical trials with the aid of pharmaceutical partners, including one for respiratory syncytial virus in Phase 2 and one for liver cancer in Phase 1. He hints that Roche and other companies soon will rue the day they decided to back out of RNAi research.

“I used to run the Boston Marathon every year, and this is like someone signing up and then quitting about 12 to 13 miles into the race,” he says. Those companies that gave up too early, he adds, “aren’t prepared to feel the thrill of the finish line.”

Promising RNAi therapeutics research also is advancing in academic labs. In March of last year, John Rossi at the Beckman Research Institute of City of Hope published a paper in Science Translational Medicine showing that attaching an aptamer to a small piece of double-stranded RNA (known as a small interfering RNA or siRNA) could provide a dual way of attacking HIV. The aptamer itself showed the ability to neutralize free-floating HIV in infected mice, and when attached to the siRNA, it ferried the siRNA into HIV-infected cells. Results showed a significantly reduced viral load in the animals treated with the combination. Rossi says the team currently is experimenting with using different siRNAs to attack multiple HIV genes at once.

He adds that Dicerna Pharmaceuticals, the RNAi therapeutics company he co-founded in 2007 based on his findings that slightly longer siRNAs than those commonly used have a more potent knock-down effect, actually got a boost while other pharma companies were pulling back. Around the time Roche announced its own RNAi program termination, Japanese pharma company Kyowa Hakko Kirin forged a $1.4 billion agreement with Dicerna.

“We’re really doing well at this point,” says Rossi, who still serves as chair of the company’s scientific advisory board.

In March of last year, chemical engineer Mark Davis of the California Institute of Technology published the results of a small Phase 1 trial of an RNAi drug targeting solid tumors aided by a nanoparticle delivery system. These encapsulated siRNAs were the right size, about 70 nanometers, to escape the leaky blood vessels that surround tumors, and they were tagged with transferin, a protein for which many cancer cells carry receptors on their surfaces. This combo allowed the siRNAs to specifically bombard tumors.

The trial showed that the therapy was safe, and biopsies from some of the volunteers’ tumors showed the RNAi was doing its job – the targeted mRNA was cleaved at just the spot where the researchers would expect. Davis and his colleagues currently are testing the therapy in a larger trial, proof that they’re not giving up anytime soon on RNAi.

“Despite what pharma says about RNAi, I think it’s a really exciting area,” he says. “I like to tell my students to work on something of high significance. It will be hard, of course, but I’d rather go down in flames working on something with high significance than something people don’t really care about.”